Protein affecting KATP channels

ABSTRACT

This invention describes the isolation and identification of a new protein, p56, useful for the identification of drugs that will selectively open or close K channels. The protein p56 has a molecular weight of about 56,000 daltons and the N-terminal peptide sequence is: Glu-Pro-Arg-Ala-Pro-Pro-Glu-Lys-Ile-Ala-Ile-Val-Gly-Ala-Gly-Ile.

FIELD OF THE INVENTION

This invention relates to potassium channels and the proteins thatcomprise those channels.

INFORMATION DISCLOSURE

The following documents are related to the cyanopyridylguanidinecompound used to isolate the protein disclosed herein.

Petersen, Hans J., et. al., "Synthesis and Hypotensive Activity ofN-Alkyl-N"-cyano-N'-pyridylguanidines," J. Med. Chem., 21, 8, pp.773-781 (1978).

U.S. Pat. No. 31,244, reissued 17 May 1983, "AntihypertensivePyridylguanidine Compounds," H. J. Petersen.

U.S. Pat. No. 4,057,636, issued 8 Nov. 1977, "AntihypertensivePyridylguanidine Compounds," H. J. Petersen.

WO 9211233-A1, published 90.12.19, "New aryl:cyano:guanidine potassiumchannel dilater--for treating hypertension," assigned to Kanebo Ltd.

European Patent 405 525 A2, published 02.01.91, "Novel CyanoguanidineDerivatives," M. Tominori.

BACKGROUND

Ionic channels of cell membranes are the basic sites where ionic fluxestake place. The modern era of the study of drug-channel interactionsbegan when voltage clamp techniques were used to demonstrate the blockof Sodium, (Na⁺), and potassium, (K⁺), channels of squid axons caused byprocaine and cocaine. Narahashi, Ann Neurology (1984); 16(suppl):S39-S51.

This invention concerns proteins which regulate or constitute the poreregion of potassium channels. Potassium channels appear to beubiquitous, found even in bacteria. See, R. Milkman, "An EscherichiaColi homologue of eukaryotic potassium channel proteins" Proc. Natl.Acad. Sci. USA, Vol 91, pp. 3510-3514, (1994). Pharmacological,biophysical and molecular studies have revealed multiple subtypes formembrane ion channels that form potassium selective pores in the plasmamembrane of many mammalian cells. One method of classifying K channelsis based on what regulates channel activity or function. For example,one class can be defined as K channels modulated by transmembranevoltage, another class modulated solely by calcium and/or nucleotides,and yet a third class modulated by G protein involvement. However, in amore simplistic manner, one can classify the family of K channels simplyby their respective gating properties. In other words, a comparison ofthe pharmacological and electrophysiological properties of potassiumchannels has given rise to an operational definition for grouping thevarious subtypes based largely on their gating properties. At present,potassium channels of known amino acid sequence comprise two distantlyrelated protein families. One of these channel families is termed,"voltage-gated," the other channel family is termed "inward rectifying."

The structure of the voltage-gated channel protein is known to becomprised of six membrane spanning domains in each subunit, each ofwhich is regulated by changes in membrane potential. B. Hille. "IonicChannels of Excitable Membranes" (Sinauer, Sunderland, Mass., 1992).Voltage-gated potassium channels sense changes in membrane potential andmove potassium ions in response to this alteration in the cell membranepotential. Molecular cloning studies on potassium channel proteins hasyielded information primarily for members of the voltage-gated family ofpotassium channels. Various genes encoding these voltage-gated family ofpotassium channel proteins have been cloned using Drosophila genesderived from both the Shaker, Shaw and Shab loci; Wei, A. et. al.,Science (1990) Vol. 248 pp. 599-603.

Unlike the voltage-gated channel proteins with six membrane spanningregions, the inward rectifier channels have only two membrane spanningdomains, each sensitive to changes in the net potassium concentration.Within this class of channels are the ATP-sensitive potassium channels.These channels are classified by their sensitivity to concentrationfluxes in ATP. The ATP-sensitive, or ATP-gated, potassium channel is animportant class of channels that links the bioenergetic situation of thecell to changes in cell function. These channels are blocked by highintracellular ATP concentrations and are open when ATP decreases.Lazdunski (1992); M. Lazdunski et al., "ATP-Sensitive K⁺ Channels",Renal Physiol. Biochem. Vol. 17: pp. 118-120 (1994).

Although ATP-gated potassium channels were originally described incardiac tissue; Noma, A. Nature (1983) Vol. 305 pp. 147-148, they havesubsequently been described in pancreatic β-cells; Cook et. al., Nature(1984) Vol. 311 pp. 271-273, vascular smooth muscle; Nelson, M. T. et.al., Am. J. Physiol. (1990) Vol. 259 pp. C3-C18 and in the thickascending limb of the kidney; Wang, W. et. al. Am. J. Physiol. (1990)Vol. 258, pp. F244-F-253.

The ATP-sensitive, or ATP-gated potassium channels play an importantrole in human physiology. The ATP-sensitive potassium channel, likeother potassium channels, selectively regulate the cell's permeabilityto potassium ions. These channels function to regulate the contractionand relaxation of the smooth muscle by opening or closing the channelsin response to the modulation of receptors or potentials on the cellmembrane. When ATP-sensitive potassium channels are opened, theincreased permeability of the cell membrane allows more potassium ionsto migrate outwardly so that the membrane potential shifts toward morenegative values. When the membrane potential shifts toward more negativevalues the opening of the voltage-dependent calcium channels is reduced,this reduces the influx of calcium ions into the cell because thecalcium channels become "increasingly less open" as the membranepotential becomes more negative. Consequently, drugs havingATP-sensitive potassium channel opening activity, drugs known aspotassium channel openers, can relax vascular smooth muscle and areuseful as hypotensive and coronary vasodilating agents. In contrast,drugs having ATP-sensitive potassium channel blocking activity, drugsknown as potassium channel blockers, inhibit ATP-sensitive potassiumchannels by decreasing potassium efflux, leading to membranedepolarization which opens voltage-gated Ca²⁺ channels. Arkhammar et al.(1987) "Inhibition of ATP-regulated K⁺ channels precedesdepolarization-induced increase in cytoplasmic free Ca²⁺ concentrationin pancreatic B-cells", J. Biol. Chem. 262: 5448-5454. These drugs findoptimal use in the stimulation of insulin secretion in type II diabetesmellitus.

A relatively large number of compounds are now known which open cellmembrane ATP-sensitive potassium channels, particularly in smoothmuscle: minoxidil sulfate, diazoxide and nicorandil are well knownpotassium channel openers. The target site for these agents ispresumably on the potassium channel itself, but may also be on anassociated regulatory protein. Isolation of the target site for thepotassium channel openers would allow for protein sequence analysis andcloning of those potassium channel opener proteins. Similar analyses ofdrug binding proteins in K_(ATP) channels have been performed for theclass of K channel blockers such as glyburide. Sulfonylurea receptorshave been analyzed on a variety of cell and tissue types using aphotoactivable form of glyburide. Aguilar-Bryan, L., et al.,"Photoaffinity Labeling and Partial Purification of the B CellSulfonylurea Receptor Using a Novel, Biologically Active GlyburideAnalog", J. Biol. Chem. (15 May 1990) Vol. 265, pp. 8218-8224.

Potassium channel openers represent a widely diverse series of compoundswhich all have the reported effect of opening only a subset of channelsdescribed as sensitive to ATP. As explained above, these compounds causephysiological responses by increasing membrane permeability topotassium, this leads to hyperpolarization of the cell membrane andtemporal desensitization to electrical and chemical stimuli.

Openers which target these channels have been synthesized as possibledrugs in hypertension, angina pectoris, coronary heart disease, asthma,and urinary incontinence. Blockers which target these channels includethe sulfonylureas, such as glyburide. The latter is an example of animportant drug which targets K_(ATP) channels in the pancreas, thusproviding a treatment for non-insulin dependent diabetes mellitus.

The rationale for the effectiveness of these drugs in targeting theK_(ATP) channel resides in the fact that this channel constitutes themain resting conductance in the B-cell. Depolarization of the channel bythe sulfonylurea blockers ultimately results in insulin release.

Despite the apparent selectivity afforded by such drugs, it also appearstrue that openers have multiple effects on target cells as well asselective effects on several tissue types. K Lawson and P. E. Hicks,"Potassium Channel Openers: Pharmacological Anomalies SuggestHeterogeneous Sites of Action", (1993) Curr. Opin. Invest. Drugs Vol 2pp. 1209-1216. It is the latter effect, that of multiple tissuetargeting, that has reduced the importance of the K channel openers asselective marketable drugs. It is essential to understand what confersselectivity of drugs to specific organs before a systematic approach canbe made towards drug design.

The membrane proteins which bind to potassium channel openers arebelieved to be structurally related, although it isn't clear whetherdrug selectivity is imparted by the channel protein itself or by thecontribution of accessory proteins. These proteins, which bind toselective drugs, may be novel K channels or they may be one of several Kchannel accessory proteins that act in concert with the primary Kchannel protein and that are needed by the system for the properphysiological response.

An analogous system using the channel blocker, glyburide, has beenexplored for pancreatic B cell K_(ATP) channels. Aguilar-Bryan, L., etal., "Co-Expression of Sulfonylurea Receptors and K_(ATP) Channels inHamster Insulinoma Tumor (HIT) Cells: Evidence for direct association ofthe receptor with the channel", J. Biol. Chem. (1992), Vol. 267 pp.14934-14940.

This invention describes the isolation and identification of a newprotein, p56, useful for the identification of selective drugs that willselectively open or close K channels. P56 is the first high affinitycyanoguanidine binding protein to be identified using a K channel openerphotoactivable probe. Unexpectedly, this opener was shown to only bindto P56 in intact cells.

SUMMARY OF THE INVENTION

This invention comprises a glycoprotein of about 54,000 to 60,000daltons and having an apparent core protein mass (free of sugars) ofabout 51,000 daltons, capable of being isolated from rat A10 cells andcapable of binding withN-(3-azido-5-iodophenyl)-N'-cyano-N"-(1,1-dimethylpropyl)-guanidine. Aglycoprotein having K_(ATP) channel activity either by itself or inmembranes with other K_(ATP) channel proteins. A glycoprotein of about56,000 daltons. A glycoprotein where the average mass of the individualsugars is about 2,500 daltons. A glycoprotein having at least threesites of N-linked glycosylation. A glycoprotein comprising the Nterminal sequence of"Glu-Pro-Arg-Ala-Pro-Pro-Glu-Lys-Ile-Ala-Ile-Val-Gly-Ala-Gly-Ile-." Aglycoprotein wherein the purified protein is the human homolog. Aglycoprotein wherein the purified protein is the murine homolog. Aglycoprotein having the characteristics of the p56 protein identifiedherein. An essentially pure glycoprotein as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Autoradiogram of a polyacrylamide gel showing the p56 protein atabout 56,000 daltons.

FIG. 2 The p56 protein after digestion with N-glycanase, endoglycosidaseH, endoglycosidase F, and a combination treatment of neuraminidase andO-glycanase.

FIGS. 3A and 3B Typical column profile of sodium dodecyl sulfateextraction of p56 applied onto a biphenyl reverse phase column.

FIG. 4 SDS PAGE (FIG. 4A) and autoradiography (FIG. 4B) of selectedfractions from a biphenyl reverse phase column.

FIG. 5 Microbore biphenyl reverse phase HPLC (Vydac) resolving thedeglycosylated p56 from N-glycanase and other contaminating A10proteins.

FIG. 6 Coomassie Blue staining of the proteins.

FIG. 7 Western blots showing the specific nature of antibody binding.

ADDITIONAL DESCRIPTION OF THE INVENTION AND DESCRIPTION OF THE PREFERREDEMBODIMENTS

The following definitions and equipment sources, are provided:

Hour is h or hr. Minute is min. Milliliters is ml. DTT isdithiothreitol; it is purchased from any of several chemical suppliers.SDS is sodium dodecyl sulfate; the electrophoresis purity reagentsupplier is Bio-Rad Laboratories, Richmond, Calif. All electrophoresisequipment was also purchased from the same vendor. NP40 is nonidet P40,a non-ionic detergent available from several chemical suppliers. HF ishydrogen fluoride/para-cresol (4-methylphenol)/para-thiocresol.

All chromatography buffers and solvents were obtained from variouschemical suppliers and were of the highest grade obtainable; all waterused in experiments was purified using a Milli-Q waterpurification/filtration system obtained from Millipore Corporation.

The YM10or YM30 membranes used for ultrafiltration concentration wereproduced exclusively by Amicon, Danvers, Mass. X-OMAT AR scientificimaging films, are made by Eastman Kodak Company, Rochester, N.Y.

Recombinant N-glycanase enzyme is obtained from Genzyme Corporation,Cambridge, Mass. The deglycosylation reactions were conducted assuggested by the manufacturer, who provide buyers a data sheet andsuggested protocol. Peptide conjugation to KLH (Keyhole LimpetHemocyanin), was conducted using the Inject Immunogen Kit obtained fromPierce, Rockford, Ill.

Electrophoresis equipment was purchased from Bio-Rad Laboratories. (ABI)476A protein sequencer (Applied Biosystems, Inc., Foster City, Calif.).PVDF solid matrix is Immobilon-P Transfer Membrane obtained fromMillipore Corporation, Bedford, Mass. SMART micropurificationchromatography system (Pharmacia LKB Biotechnology, Uppsala, Sweden);chromatography columns used with this system are available throughseveral manufacturers.

Amino acid residues referred to in this application are listed below,they may also be given either three letter or single letterabbreviations, as follows:

Alanine, Ala, A; Arginine, Arg, R; Asparagine, Asn, N; Aspartic acid,Asp, D; Cystein, Cys, C; Glutamine, Gln, Q; Glutamic Acid, Glu, E;Glycine, Gly, G; Histidine, His, H; Isoleucine, Ile, I; Leucine, Leu, L;Lysine, Lys, K; Methionine, Met, M; Phenylalanine, Phe, F; Proline, Pro,P; Serine, Ser, S; Threonine, Thr, T; Tryptophan, Trp, W; Tyrosine, Tyr,Y; Valine, Val, V; Aspartic acid or Asparagine, Asx, B; Glutamic acid orGlutamine, Glx, Z; Any amino acid, Xaa, X.

All amino acids have a carboxyl group and an amino group. The aminogroup of the amino acid is also referred to as the "N-terminus" of theamino acid. The carboxyl group of an amino acid is also referred to asthe "C-terminus" of the amino acid. The "N-terminus" of an amino acidmay form a peptide bond with a carboxyl group of another compound. Thecarboxyl group that combines with the "N-terminus" of an amino acid maybe the carboxyl group of another amino acid or it may be from anothersource. If several amino acids are linked into a polypeptide, then thepolypeptide will have a "free" N-terminus and a "free" C-terminus.

The materials and methods used to isolate, identify and characterize theprotein are provided.

Materials. The cyanoguanidine,N-(3-azido-5-iodophenyl)-N'-cyano-N"-(1,1-dimethylpropyl)-guanidine, a Kchannel opener utilized for photoaffinity labelling, is synthesized. Thesynthesis is according to the procedures described in U.S. patentapplication Ser. No. 98/257856, Gadwood, et. al.,Azidophenylcyanoguanidine and Their Use as Photoaffinity Probes. Thedesignated compound contained an aryl azide radiolabelled with ¹²⁵ I! toa specific activity of 2200 Ci/mmole. (New England Nuclear).

Electrophoresis. One-dimensional analytical SDS polyacrylamide gelelectrophoresis is conducted using 10% gels in a mini Protean II system(Bio-Rad Laboratories) according to the method of Laemmli, U. K.Laemmli, (1970) Nature, Vol. 227, pp. 680-685, adjusted with minormodifications. Before electrophoresis samples are diluted 1:1 withdenaturation buffer (2% SDS, 25% glycerol, 0.25M Tris HCl, pH 6.8, and1% β-mercaptoethanol). Electrophoresis is conducted at constant power (5watts/gel) for 1 hour at room temperature and terminated when the dyefront (bromphenol blue) reaches the bottom of the gel. The completedgels are either electroblotted onto nitrocellulose or PVDF, or fixed in50% ethanol and 10% acetic acid, and stained with Coomassie BrilliantBlue G-250.

Analysis of Radiolabelled Proteins (Assay Method). Radiolabelledfractions may be analyzed in several ways depending on the state of thesample. For solution samples, simply count a portion or all of thesample in a gamma counter (0.5 to 1.0 min per sample). For wet gels,incubate the gels in a -70° C. freezer with XOMAT AR x-ray film, or cutthe gel into 2 mm pieces and count the sections in a gamma counter. Fordried gels or dried blot papers, a phosphorimager (Molecular Dynamics)for quantitation of the individual bands may be used.

A10 Cell Growth, Photoaffinity Labelling, Membrane Preparation. A10 celllines derived from embryonic rat aorta are obtained from the AmericanType Culture Collection (CRL-1476). The cells are subcultured and grownto confluence in Corning 150 mm tissue culture plates at 37° C. in 6.0%CO₂ in Dulbecco's Modified Eagle's Medium supplemented with 20% (v/v)fetal bovine serum. The cells are then washed 2X with Earle's balancedsalt solution buffered to pH 7.4 with 20 mM HEPES (EBSS-H). The cellsare placed in EBSS-H containing 10 nM ¹²⁵ I!-U-97149. The A10 cells areequilibrated with the radiolabeled compound for 15 min at 37° C. The A10cells are then placed on ice for 2 min and exposed to 600 μwatts/cm² of254 nm UV light.

After photolysis, the A10 cells are washed extensively with phosphatebuffered saline. Membranes from the A10 cells are solubilized in acocktail of 0.2% Triton X-100 detergent and 20 mM Tris pH 6.8 andprotease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/mlpepstatin, and 5 mM benzamidine). The membranes are precipitated with 4volumes of ice cold acetone for 60 min on dry ice. The precipitatedmembrane proteins are collected by centrifugation at 20,000 rpm for 30min at 4° C. in a Beckman SW-28 rotor. After centrifugation thesupernatant is discarded and the pellet is dissolved in 1% SDS and 10 mMβME.

Purification of the protein p56. Preparative SDS PAGE--Tritonsolubilized ¹²⁵ I!-CG-labelled A10 membrane protein preparations from24-48 large culture plates are acetone precipitated and washed. Theresulting pellets are resolubilized in 1% sodium dodecyl sulfate (SDS)containing 10 mM dithiothreitol (DTT) (approximately 6-12 ml finalvolume) as described above. An equal volume of sample denaturationbuffer is added (125 mM Tris, HCl pH 6.8, 1% SDS, 10 mM DTT), and aftermild heating and mixing, the sample is distributed onto 4 preparativepolyacrylamide gels (10% total; 37.5:1.0 acrylamide to bis-acrylamideratio; 1.5 mm thickness). These gels are run according to the method ofLaemmli with minor modifications in a Protean II cell at low voltage(25-30 V limited) overnight at room temperature. The upper tank bufferis supplemented with 1 mM sodium thioglycollate to reduce possibleprotein modification due to oxidation or free radicals within the geland to keep the proteins in reduced form during the run to preventdisulfide linked aggregation.

Upon completion of electrophoresis, both the dye front, which consistsmainly of free drug, and one of the major proteins, actin, arevisualized by incubation of the completed gels in a solution of 0.1Mpotassium chloride for about 5 min. Following brief washings indeionized water, the position of actin is marked and the bottom of thegel is cut to allow for recognition patterns on developingautoradiograms. Each gel is wrapped in saran wrap and sealed in plasticbags. Autoradiograms are made after 2-3 h exposure (-70° C.) of the wet,frozen gels to X-OMAT AR films. These autoradiograms are then used as aguide for the excision of the target protein band; in this case, p56.The excised polyacrylamide gel strips are minced and fragmented using amortar and pestle, and incubated in a large volume of 1% SDS containing10 mM DTT (˜500 ml). After a 2 hour incubation with stirring, thesolution is centrifuged at low speed in 50 ml tubes for 5 min. at roomtemperature. The supernatant is removed and poured through a 0.22 micronfilter to remove remaining polyacrylamide fines. The resulting solutionis concentrated by Amicon ultrafiltration (YM-30 membrane) to a finalvolume between 6 and 12 ml.

Reverse Phase Chromatography. The concentrate from the above preparativeSDS PAGE extract of p56 is injected onto a 0.46×15 cm (10μ) biphenylHPLC column. The column profile is developed with a gradient of 32% to54.4% acetonitrile in 0.1% triflouroacetic acid over a period of 40 minat a flow rate of 1 ml/min (1 ml fractions are collected). Aradioactivity profile is obtained of individual fractions by gammacounting at 0.5 min each for ¹²⁵ I!. Appropriate fractions representingp56 as judged by SDS PAGE and blotting, followed by phosphorimaging toconclusively show the location of p56 in the resolved fractions, arepooled and dried by vacuum centrifugation (SpeedVacEvaporator/Concentrator System, Savant Instruments, Inc., Farmingdale,N.Y.).

Deglycosylation of p56. The biphenyl reverse phase HPLC purified p56sample is redissolved in minimal 1% SDS, and heated in boiling water for2 min. After heating, the sample is treated with the non-ionicdetergent, NP-40, such that for each 1% of SDS, a minimum of 1.5% ofNP-40 is added. This solution is incubated in sodium phosphate, pH 7.0,and is mixed with N-glycanase (Genzyme) as suggested by themanufacturer. The deglycosylation is allowed to proceed overnight at 37°C.

Microbore (SMART) HPLC. In some instances, samples after deglycosylationare further resolved on a microbore biphenyl column (2.7 mm×15 cm) on aSMART system (Pharmacia-LKB) at 100 μl/min.

Electroblotting. Transferral of proteins from SDS gel to Immobilon-PTransfer Membrane (PVDF; Millipore Corp., Bedford, Mass.) is performedwith a semi-dry blotter at -15 mA/cm² for 15 min;

Sequence analysis. N-terminal sequencing of the deglycosylated p56protein electrophoretically transferred to PVDF, see, P. Matsudaira(1987) J. Biol. Chem. Vol. 262 pp. 10035-10038, after purification bySDS-PAGE, is performed on an Applied Biosystems Inc. (ABI) Model 476Aprotein sequencer.

Synthesis of the N-Terminal Peptide. Solid phase peptide synthesis(Barany & Merrifield, 1979, in The Peptides, Vol. 2, pp. 1-284, E. Grossand J. Meienhofer, editors, Academic Press, New York) is performed at0.5 mmole scale utilizing Boc-Cys(4-CH₃ Bzl)OCH₂ Pam resin (AppliedBiosystems Inc., Foster City, Calif.) on an Applied Biosystems Inc. 430APeptide Synthesizer. Amino acids may be obtained from any commerciallyavailable source. In the examples shown here all amino acids wereobtained from Applied Biosystems Inc. The t-butyloxycarbonyl (BOC) groupis used as the N-amino protecting group during step-wise synthesis.Tri-functional amino acid side chains are protected as follows:Arg(Tos), Glu(OBzl), and Lys(Cl--z). Each residue is coupled twice, thencapped with acetic anhydride before the next cycle of synthesis.Quantitative ninhydrin tests are performed at each cycle of thesynthesis. After removing the N-terminal Boc group in the usual fashion,the peptide is cleaved from the resin by treatment with Hydrofluoricacid (HF)/p-cresol/p-thiocresol (10:0.5:0.5) for 1 hour at -200°to -5°C.

The peptide resin is titrated with ether, the crude peptide dissolved in50% acetic acid and the resin removed by filtration. The filtrate isevaporated to dryness under reduced pressure and lyophilized fromglacial acetic acid. The crude peptide is purified by preparativereverse phase chromatography on a Vydac C-18 column (250×22.5 mm) usinga water acetonitrile gradient, with each phase containing 0.1% TFA.Clean fractions, as determined by analytical HPLC, are pooled andacetonitrile evaporated under reduced pressure; an aqueous solution ofthe pooled fractions is lyophilized. The purified peptide ischaracterized by time of flight mass spectroscopy. The anticipated(M+H)+ is 1878.9.

Preparation of N-Terminal p56 Polyclonal Antibody. The N-terminalpeptide, prepared above, is conjugated to KLH-maleimide (Pierce ChemicalCo.), using procedures supplied by the manufacturer, to form theKLH-peptide conjugate at a final concentration of 4.0 mg/ml (KLH) and2.7 mg/ml (peptide). Verification of coupling is made using Ellman'sreagent. The KLH-peptide conjugate is separated from the free peptide bydialysis versus 1X PBS, pH 7.5. At the same time, a peptide conjugatedto ovalbumin is prepared in an identical manner to provide for a samplewhich could be used to screen the test and production bleeds as they areproduced. For the latter, the peptide-ovalbumin conjugate exhibited anapparent molecular weight of 60-65,000, compared to 45,000 for theunconjugated ovalbumin protein.

To test whether the rabbit sera is immunoreactive with the peptide,Western blots are conducted on nitrocellulose strips containing theovalbumin-peptide conjugates at various concentrations. The primary seraare tested at a 1:100 dilution in TN buffer (20 mM Tris HCl, pH 7.5,0.5M NaCI) containing 1% BSA as carrier. The secondary antibody consistsof dilute solutions of alkaline phosphatase-conjugates ofgoat-anti-rabbit IgG in TN buffer, supplemented with 1% BSA as carrier.Positives on the blots are visualized using AP substrates, NBT(p-nitroblue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3-indolylphosphate). To test whether the sera are capable of detecting p56 onblots, subject crude, partially purified and purified p56 samples to SDSpolyacrylamide gel electrophoresis (10%), and blot onto nitrocellulose,as defined above. The blots are then probed using primary sera which hadeither been pre-adsorbed with the free N-terminal synthetic peptide orleft unchallenged.

The following documents, incorporated by reference, also contain usefulmethods generally known to one skilled in the art: U. K. Laemmli (1970)Nature 227:680-685 and P. Matsudaira (1987) J. Biol. Chem.262:10035-10038.

UTILITY OF THE INVENTION

This invention describes the isolation and identification of a newprotein, p56, useful for the-identification of drugs that willselectively open or close K channels. The p56 protein is the first highaffinity cyanoguanidine binding protein to be identified using a Kchannel opener photoactivable probe. Unexpectedly, this opener was shownto only bind to p56 in intact cells, supporting the role of this proteinin native potassium channel activity.

The p56 protein is likely to be a K_(ATP) channel or an accessoryprotein that regulates K_(ATP) channel activity. As an accessory proteinin the channel, it would likely impart selectivity and specificitytowards binding of potassium channel directed drug molecules. In eithercase, as a channel or accessory to a channel, p56 is an important andnovel drug target.

The identification of a larger portion of the amino acid sequence willlead to the design of oligonucleotide probes which will permit thecloning of p56 from various species and expression of p56 protein inbacterial and mammalian cell systems. An analogous approach has beentaken by Bryan et al. in the isolation, characterization, and cloning ofthe glyburide receptor in HIT cells. Bryan, J., Aguilar-Bryan, L., andNelson, D., "Cloning of a Sulfonylurea Receptor (ATP-Sensitive K⁺Channel ?) from Rodent a- and B- Cells, First International Conferenceon ATP-Sensitive K⁺ Channels and Sulfonylurea Receptors (Sep. 30-Oct. 1,1993), Houston, Tex., pp. 149-153.

Knowledge of the amino acid sequence for p56 will allow the design ofappropriate oligonucleotide probes for determination of mRNA levels incell and tissue preparations using in situ hybridization experiments.Knowledge of the sequence will also allow the examination of thestructure of the protein by computational software programs, providing adirect method for primary and secondary structure comparison of p56 toknown potassium channel proteins.

The identification of this protein will allow the design of higheraffinity polyclonal antibodies and/or monoclonal antibodies to bedeveloped that recognize p56 in different species. Such antibodies willallow cell and tissue distribution of the protein to be determined.Antibodies to the p56 protein will allow for immunocytochemistry andhistological examination of p56 protein expression in cells and tissuesections to complement the analysis of mRNA levels by Northern blotanalysis.

This system can be used to study how potassium channel openers andblockers interact with the channel complex by competition studies, andto study and identify the other members of the potassium channelcomplex.

Additional understanding of the utility of the invention can be found inthe following documents, incorporated by reference: U. Quast (1993) "Dothe K+ Channel Openers Relax Smooth Muscle by Opening K+ Channels ?",Trends In Pharmaceutical Sciences 14:332-337. U. Quast, K. M. Bray, H.Andres, P. W. Manley, Y. Baumlin, and J. Dosogne (1993) Binding of theK+ Channel Opener, ³ H!P1075 in rat isolated aorta: Relationship tofunctional effects of openers and blockers. Molecular Pharmacology43:474-481. D. R. Howlett and S. D. Longman (1992) Identification of abinding site for ³ H! cromakalim in vascular and bronchial smooth musclecells. British J. Pharmacol. 107:396P. Barany, Merrifield (1979) in ThePeptides (Gross, E., and Meienhofer, J., eds.), Vol. 2, pp.1-284,Academic press, New York.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have isolated and purified a unique protein calledp56. This protein is either a true K channel protein or an accessoryprotein which might confer selectivity to a given channel. TheN-terminus of the protein has been determined and a peptide representingthe N-terminus of the N-deglycosylated p56 was synthesized. Polyclonalantibodies to the peptide were created which immunoreact with both thefree peptide as well as authentic p56 protein.

Radiochemically labelled and photoactivable K channel openers are usedto identify cyanoguanidine binding proteins in a membrane preparation ofA10 cells. One suitable probe isN-(3-azido-5-iodophenyl)-N'-cyano-N"-(1,1-dimethylpropyl)-guanidine, seeformula 1, below. ##STR1##

The proteins in the A10 cell preparation are first photolabelled, andthen the cells are extracted with a cocktail of 0.2% Triton X-100detergent and 20 mM Tris HCl, pH 6.8, containing protease inhibitors (10ug/ml leupeptin, 10 ug/ml aprotinin, 10 ug/ml pepstatin, and 5 mMbenzamidine). A cold solution of acetone is added to the Triton extractto precipitate the proteins, and allow for the removal of the unreactedphotoaffinity label in the supernatant. Proteins present in the acetonepellet, including those radiolabelled proteins which were photolabelled,were then extracted with 1% sodium dodecyl sulfate (SDS) containing 10mM dithiothreitol or 10 mM 2-mercaptoethanol.

Preliminary studies of the protein, p56, showed that the solubility ofthe protein was lost in the absence of SDS. However, use of SDSsolutions of crude membrane proteins was found ineffective forpurification using several reverse phase columns (C4 or C18). A commonproblem was the resolution of entire micellar products containingseveral different sized proteins, without significant purificationafforded. To avoid these problems, we chose a biphenyl HPLCchromatography step, since this column accommodates solutions of 1% SDS,allows for resolution of SDS-dissolved proteins, and doesn't result inbroadening effects noted in C4 and C18 column profiles.

Therefore, since the presence of SDS in the sample could be toleratedprior to HPLC resolution, we preferred to first select for the sizerange of proteins which are approximately 56 kilodaltons by preparativeSDS PAGE as described in the Methods section. This method effectivelyremoves the contaminating radiolabelled proteins which are either loweror higher molecular weight than p56. A resulting autoradiogram of atypical wet gel after 2 hours incubation with X ray film at -70° C.shows a complex pattern of radiolabelled proteins, with a major labelledprotein easily detected at 56,000 daltons, see FIG. 1. The Y-axis ofthis illustration depicts the molecular weight, with the bottomrepresenting the location where the smallest proteins migrate and thetop representing the location where the largest proteins migrate (therelationship of migration distance to molecular weight is a logarithmicfunction). For these studies we commonly utilized 10% polyacrylamide(37.5:1.0 acrylamide:bis-acrylamide), resulting in an effective range ofseparation of proteins having masses from 15 kilodaltons through 200kilodaltons.

The relative molecular weight of p56 was determined by comparison of itsmigration to that of a series of proteins having known masses. Thepredicted mass is determined following a linear regression analysis ofthe migration distances of the known standard proteins versus theirmolecular masses. When the mass of p56 is calculated by this method, anaverage mass of 56 kD is measured, with variability of the measurementlimiting the size from 54 to 60 kD.

FIG. 1 shows that there are several radiolabelled bands present on thegel making it difficult to discern specific from non-specificradiolabelling. However, we know that the p56 band is specific sinceexcess cold drug competition results in loss of detectable radioactivityat this position (data not shown). The major band noted at 56,000daltons on the gel (as located by the autoradiogram) was excised andextracted out by passive diffusion in a 1% solution of sodium dodecylsulfate containing 10 mM dithiothreitol. The resulting solution wasconcentrated to a small volume (less than 8 ml) by ultrafiltration(Amicon Corporation, Lexington, Mass.).

A determination of whether p56 is a glycoprotein was made by digestingthe SDS extracted protein solution with N-glycanase, endoglycosidase H,endoglycosidase F, and a combination treatment of neuraminidase andO-glyeanase. The results of experiments with a partially purifiedpreparation of ¹²⁵ I! p56 using these enzymes are shown in FIG. 2. FIG.2 shows the results of digestion of p56 (left to right) with:N-glycanase (Lane 1), O-glycanase+Neuranminidase (Lane 2), Endo H (Lane3), Endo F (Lane 4), and Control (Lane 5).

Results show that p56 is sensitive only to Endo H and N-glyeanase (asjudged by a demonstrated change in migration of the radiolabelled bandon the gel), suggesting that the protein contains one or more sites ofN-linked glycosylation of a high mannose type. The negative results withneuraminidase and O-glycanase suggested no O-linked glycosylation orsialic acid residues present in the glycoprotein.

The deglycosylation pattern of p56 was examined with variableconcentrations of N-glycanase. Analysis indicates that in addition top56 and its fully deglycosylated product (p52), there are at least twointermediate forms of glycosylated p56. We thus conclude that there areat least three sites of N-linked glycosylation on the p56 proteinisolated from A10 membranes.

Purification and Characterization of p56.

Starting with a concentrate of the sodium dodecyl sulfate extraction ofp56 from gel slices, radiolabelled as a consequence of reacting with thephotoactivatable K channel opener, as explained above, the sample isapplied onto a biphenyl reverse phase column.

A typical column profile is shown in FIGS. 3A and 3B. FIG. 3A shows theradioactivity profile (X axis is retention time as column fractions; Yaxis=CPM), and the profile of the absorbance of the sample is shown inFIG. 3B (X axis=Retention Time; Y axis=Absorbance at 215 nm A₂₁₅ !). Thelatter absorbance represents the most sensitive region of the absorptionprofile of proteins, indicative of peptide bonds. An examination of theselected fractions from this step by SDS PAGE is shown in FIGS. 4A and4B. FIG. 4A shows the stained polyacrylamide gel, while an autoradiogramof the same gel is shown in FIG. 3B. The data shows the protein andradiochemical and protein purity, respectively, at this stage of thepurification process. The p56 protein, defined by radioactivity andsize, is noted in fractions 33-41. Appropriate fractions are selectedand dried by vacuum centrifugation. The dried sample is redissolved inSDS containing buffer and subjected to deglycosylation with N-glycanase.The product of this step results in a p56 protein which is now devoid ofN-linked oligosaccharides.

The pool is then subjected to microbore biphenyl reverse phase HPLC(Vydac) to resolve the deglycosylated p56 from N-glycanase and othercontaminating A10 proteins, as shown in FIG. 5. FIG. 5 shows theabsorbance (unshaded) and the radioactivity (shaded) of the fractions (Yaxis) versus the retention time (X axis) resulting from this final HPLCresolution step. This figure shows that additional resolution of p56 isobtained since the net radioactivity (fractions 25-32, shaded plot) isresolved from the major contaminating proteins (depicted by absorbanceat 215 nm, unshaded plot). Individual fractions containingdeglycosylated p56 (same figure, fractions 25-32) were subjected to SDSPAGE and blotted onto PVDF. The results of this experiment are shownfollowing staining the proteins on the blot with Coomassie BrilliantBlue R-250, see FIG. 6. The arrow in the figure depicts the location ofdeglycosylated p56, whose identity was confirmed by detection ofradioactivity using phosphorimaging (data not shown). To prepare thesegment of the blot containing deglycosylated p56 for microsequencing,the section representing the radiolabelled band was cut out of the PVDFpaper. To verify that the band was indeed excised correctly, theremaining PVDF paper was reanalyzed by phosphorimaging to confirm thatthe radioactive band had indeed been selected precisely.

N-Terminal Sequence Analysis of p56.

Following deglycosylation and PVDF blotting, see P. Matsudaira (1987) J.Biol. Chem. Vol. 262, pp. 10035-10038, a peptide sequence was obtained(Glu-Pro-Arg-Ala-Pro-Pro-Glu-Lys-Ile-Ala-Ile-Val-Gly-Ala-Gly-Ile-) Seq.ID. NO. 1, for a sample that was clearly in the picomolar range, seeTable I below for yields per sequencing cycle. Thus, the amount ofprotein which was sequenced is estimated at 0.68 picomoles (from aminimum of 48 plates of A10 cells) based on the first cycle ofsequencing. For a protein having a molecular mass of 52,000 daltons,this represents a yield of 0.7 nanograms from each plate of A10 cells.This value for the protein yield is based on the accumulation of allpurification steps, using as an assay the radiolabelled p56 protein, forwhich an efficiency of labelling by the photoactivatable cyanoguanidinewas estimated at 0.05%. The yield, then, does not necessarily representthe actual amount of p56 expressed in A10 cells.

The peptide sequence obtained is not only the putative amino terminal ofp56, but also a "unique" sequence, not observed in protein sequencedatabases. In a general search of proteins showing identity to the p56N-terminal peptide, no homology was noted to any mammalian potassiumchannel protein. A polyclonal antibody against the N-terminus of theprotein was created to verify the conclusion that the sequence of thepolypeptide was the same as the sequence of the N-terminus of theprotein. Table I, next page,

                  TABLE I    ______________________________________    N-Terminal Sequencing of Deglycosylated p56    Residue #    Amino Acid                           Quantity (pmoles)    ______________________________________    1            Glu       0.68    2            Pro       0.39    3            Arg       1.50    4            Ala       1.40    5            Pro       1.46    6            Pro       1.54    7            Glu       0.93    8            Lys       0.46    9            Ile       1.06    10           Ala       1.29    11           Ile       1.14    12           Val       1.60    13           Gly       1.13    14           Ala       1.11    15           Gly       1.02    16           Ile       0.62    ______________________________________

Development of a Polyclonal Antibody to the N-Terminus of p56.

The peptide representing the N-terminus of the N-deglycosylated p56 wassynthesized, EPRAPPEKIAIVGGC SEQ. ID. NO. 2, (see Formula 2 below),terminal GGC added to aid conjugation to KLH, the sequence without theterminal GGC is SEQ. ID. NO. 3, and used for immunization of a singlerabbit.

    H-Glu-Pro-Arg-Ala-Pro-Pro-Glu-Lys-lle-Ala-lle-Val-Gly-Gly-Cys-OH

    3 CF.sub.3 --COOH

Formula 2

The first antigen dose was administered subcutaneously with Freundscomplete adjuvant. After three weeks, an observable titer against thepeptide (as measured by peptide conjugated to ovalbumin) was detected,although no response to the p56 protein was noted by Western blotting atany dilution of the serum. At this point, the antigen was administeredsubcutaneously with Freunds incomplete adjuvant, a process continued forat least half a year.

After the second bleed (6 weeks), a response was detected against p56 onWestern blots (using partially purified as well as crude lysatematerial). Subsequently, all production bleeds of this single rabbithave yielded antibody with high titers against rat A10 p56. We havetermed this serum antibody, which is defined as containing one or moreIgG's specific for the N-terminal 12 amino acids of rat A10 p56, UP76.

Importantly, the bands immunodetected on blots were exactly coincidentwith the radioactivity profile noted by phosphorimaging. To demonstratethat the peptide sequence was derived from p56, purified samples of p56were deglycosylated with N-glycanase and analyzed by Western blotting.Conclusively, both before and after deglycosylation, the radiolabelledprotein was exactly coincident with the immunodetected band. Thisobservation eliminated the possibility that the sequence obtained wasfrom a 50-52 kD contaminating protein rather than from p56 itself.

To verify that the bands detected on Western blots were not due to anon-specific binding phenomenon, the response on blots was blocked bypre-incubation of the serum with the peptide as shown in FIG. 7. ThisFigure shows two Western blots of gels containing resolvedphotoaffinity-labelled crude A10 proteins. The left figure shows theresults of probing one of the two duplicate blots with the anti-p56serum. In this result one detects the presence of bands at 56 kD, at 30kD, and at 35 kD. The right figure shows the results of probing one ofthe blots with peptide-competed anti-p56 serum. An analysis of thelatter blot probed with peptide pre-adsorbed serum shows that the 56 and30 kD bands are definitively peptide competed (since they are no longerdetected on the blot), and thus contain the sequence shown in Formula 2.The band at 35 kD is not related to p56 since it is not competed bypeptide (Formula 2), and is therefore the result of a non-specificinteraction of the antibody with this unknown antigen. The above dataindicate that the band at 30 kD, which contains an epitope which iscompeted with peptide (Formula 2), probably originated either throughproteolytic truncation of p56 or as a separate gene product.

The N-terminus of p56 is species-specific since no cross-reactivity isobserved, using the rabbit anti-rat p56 antibody defined above, versusmurine p56. A murine cell line derived from brain smooth muscle waslabelled with the photoprobe. When the solubilized membrane pool wasexamined by Western blotting, no band was observed at p56. Similarly,following dissection of various tissues from a mouse, Western blottingwas used to locate p56. Again, no signal was detected for non-rat p56samples. In an analogous manner, COS cell extracts also failed toexhibit a p56 protein which cross-reacts with the UP76 antibody.However, photolabelling of murine brain smooth muscle cells withN-(3-azido-5-iodophenyl)-N'-cyano-N"-(1,1-dimethylpropyl)-guanidineindicated the presence of a radiolabelled band in the p56 area. Thissuggests the N-terminus of p56 is species-specific as reflected byWestern blotting with UP76, but that p56 is probably present in otherspecies as suggested by the results of photoaffinity labelling.

The p56 protein is present in the kidney, brain, trachea, and pancreasof rats. Utilizing the UP76 polyclonal antibody, described above, theextracts of various tissues from a dissected rat were examined byWestern blotting. Both the soluble pools as well as the membrane pools(detergent solubilized) were used for these experiments. By Westernblotting, bands at 56 kD and 30 kD were observed for pancreas, brain,trachea and kidney, each of which was eliminated when the serum had beenpreadsorbed with the competing peptide, shown in Formula 2.

These data demonstrate, for the first time, the presence of p56 intissues other than aortic smooth muscle. The presence of p56 in thesetissues was also established by examination of the sensitivity of thep56 bands to deglycosylation with N-glycanase. In all cases, a shiftfrom p56 to p52 was observed as expected and as noted for authentic p56from A10 cell membranes. Immunoreactive p56 was also observed incommercially obtained frozen rat kidney and brain tissue extracts.

We have also purified the specific IgG fraction from the crude serum ofUP76, the rabbit anti-rat p56 antibody using an immobilized form of thepeptide antigen, (see Formula 2). Following binding of the total IgGpool to Protein A-Agarose, the IgG was eluted by dissociation at low pH.Following adjustment of the pH to neutrality, the IgG pool was incubatedwith an immobilized form of the peptide (formula 2). Following extensivewashing to remove undesired IgG proteins, the specific peptide-bindingIgG was eluted by low pH dissociation, neutralized to pH 7.5, andconcentrated by ultrafiltration (Amicon). This specific antibody isbeing used for expression cloning attempts and to determine tissue andcell specificity.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 3    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GluProArgAlaProProGluLysIleAlaIleValGlyAlaGlyIle    151015    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GluProArgAlaProProGluLysIleAlaIleValGlyGlyCys    151015    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GluProArgAlaProProGluLysIleAlaIleVal    1510    __________________________________________________________________________

We claim:
 1. A glycoprotein of about 54,000 to 60,000 daltons, asmeasured by SDS PAGE gel and having a core protein mass (free of sugars)of about 51,000 daltons, as measured by SDS PAGE gel purified from ratA10 cells and capable of binding withN-(3-azido-5-iodophenyl)-N'-cyano-N"(1,1-dimethylproply)-guanidine.
 2. Aglycoprotein of claim 1 having K_(ATP) channel activity either by itselfor in membranes with other K_(ATP) channel proteins.
 3. A glycoproteinof claim 1 of about 56,000 daltons, as measured by SDS PAGE gel.
 4. Aglycoprotein of claim 3 where the mass of the individual sugars of theglycoprotein, as measured by SDS PAGE gel is about 2,500 daltons.
 5. Aglycoprotein of claim 4 having at least three sites of N-linkedglycosylation.
 6. A glycoprotein of claim 5 comprising the N terminalsequence of SEQ. ID. NO.
 1. 7. A glycoprotein of about 54,000 to 60,000daltons, as measured by SDS PAGE gel and having a core protein mass(free of sugars) of about 51,000 daltons, as measured by SDS PAGE gelpurified from murine cells and capable of binding withN-(3azido-5-iodophenyl)-N'-cyano-N"(1,1-dimethylproply)-guanidine.
 8. Aglycoprotein of claim 7 having K_(ATP) channel activity either by itselfor in membranes with other K_(ATP) channels.
 9. A glycoprotein of claim7, as measured by SDS PAGE gel of about 56,000 daltons.
 10. Aglycoprotein of claim 9, where the average mass of the individual sugarsof the glycoprotein, as measured by SDS PAGE gel is about 2,500 daltons.11. A glycoprotein of claim 10, having at least three sites of N-linkedglycosylation.
 12. A glycoprotein of claim 11, comprising the N terminalsequence of sequence ID NO.1.